(M0930-09-54) Quantitative Expression of Stemness Markers to Identify Metastatic Potential of Triple-Negative Breast Cancer Cells: Prognostic Signature for Personalized Treatment

Purpose: Triple-negative breast cancer has a higher risk of metastasis and tumor recurrence. Cell-specific quantitative screening approach would advance the predictive analysis of metastatic potential as an effective tool in cancer management. This study aims to quantitate stemness marker expression to identify the relative metastatic potential of triple-negative breast cancer cells. Furthermore, we aim to understand the prognostic signatures of altered stemness marker expression concerning the metastatic potential of anticancer drug-treated cells. Eventually, we will decipher the significance of combination anticancer drug treatment to reduce Doxorubicin-induced cell stemness.

Methods: A cross-sectional study was conducted using MDA-MB 231 and human triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) cells (tissues obtained from Sanford Broadway Clinic). PDX model for TNBC cells was conducted using female NSG mice (NOD SCID) (IACUC-approved protocol number A2105). Stemness markers, CD24 and CD133 expressions were quantitated in normoxic and hypoxic conditions for MDA-MB 231 and TNBC PDX cells using immunofluorescence confocal microscopy images and ImageJ macro (Fig. 1A and 1B). In addition, the cell invasion assay was conducted on the MDA-MB 231 and TNBC PDX cells using 0.4 mm polycarbonate membrane inserts. The invaded cells were considered to have high metastatic potential, and the expression of stemness markers was correlated with the noninvaded cells (Fig. 2A). The altered CD24 and CD133 expression accounted under Doxorubicin (Dox) and All-trans retinoic acid (ATRA) combination drug treatment. TNBC PDX cells were exposed to Dox and ATRA at synergistic concentration. Enhanced CD24 and CD133 expression of Dox-treated cells were termed Dox-induced cell stemness and altered Dox-induced cell stemness expression was accounted for in response to Dox and ATRA combination treatment.

Results: Quantitative evaluation of CD24 and CD133 expression revealed diverse distribution in tumor cells under normoxic and hypoxic conditions and Dox and ATRA-treated cells. The confocal microscopy images indicate enhanced CD24 and CD133 expression in hypoxic conditions compared to normoxic conditions for MDA-MB 231 and TNBC PDX cells (Fig. 1C and 1D). Compared to normoxic conditions, in hypoxia, mean CD24 expression was higher by ~25.9 folds and ~3.1 folds in MDA-MB 231 and TNBC PDX cells, respectively, as well as CD133 expression was higher by ~7.6 folds and ~7.8 folds in MDA-MB 231 and TNBC PDX cells, respectively (Fig. 1E and 1F). Invaded TNBC PDX cells in hypoxic conditions demonstrated ~2.4 folds and ~9 folds higher CD24 and CD133 expression, respectively, compared to noninvaded cells. However, in normoxic conditions, wide distribution of CD24 (normalized intensity: ~8 to ~110) and CD133 (normalized intensity: ~5 to ~98) expression was observed for invaded tumor cells (Fig. 2B and 2C). The confocal microscopy images of invaded TNBC PDX cells show enhanced CD24 and CD133 expression in hypoxic conditions compared to normoxic conditions (Fig. 2D). Dox-induced enhanced CD24 and CD133 expression were observed in both normoxic and hypoxic conditions (Fig. 3A). Dox-induced enhanced CD24 and CD133 were more prominent in hypoxic conditions and noticed up to ~4.5-fold Dox-induced enhanced expression. Dox and ATRA demonstrated a synergistic effect at specific drug concentrations. For Dox and ATRA combination treatment with synergistic concentrations, we observed a notable trend toward reduced Dox-induced CD24 and CD133 expression (Fig. 3B).

Conclusion: Variation of stemness marker expression suggests the significance of personalized treatment and represents the heterogeneous nature of tumor cells. In hypoxic conditions, enhanced stemness marker expression of invaded cells suggested a positive correlation of stemness marker expression for metastatic potential of cancer cells. Hypoxia plays a significant role in predicting altered stemness marker expression of TNBC cells. Further Dox-induced enhanced stemness marker explains a higher risk of tumor recurrence post-chemotherapy. DOX and ATRA combination treatment suggest synergistic anticancer effect reducing Dox-induced enhanced stemness expression. This study strongly implicates a perspective utility of screening stemness marker of TNBC tumor cells as a prognostic signature for personalized treatment to identify the metastatic potential of tumor cells and is highly imperative to clinical decisions in cancer management.

Acknowledgements: The NIH grant 2 R01GM 114080, NSF EPSCoR Track-1 Cooperative Agreement OIA #1946202, and DaCCoTA Center award (NIGMS U54 GM128729) supported this study. We would also like to acknowledge the core confocal microscopy facility, Department of Pharmaceutical Science, NDSU supported under the Dakota cancer collaborative on translational activity (DaCCoTa CTR) (NIH/NIGMS: U54GM128729). Any opinions, findings, conclusions, or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the NSF.


Fig. 1. Triple-negative breast cancer (TNBC) cells express stemness markers distinctly in a hypoxic microenvironment. TNBCs (MDA-MB 231 and TNBC patient-derived xenograft cells) were cultured for four days in a normoxic and hypoxic environment, fixed, stained with DAPI, phalloidin, cytoplasmic dye, and either CD24-AF647 Ab or CD133-AF-647 Ab and imaged by using Airyscan Confocal laser-scanning Microscopy (AC-LSM). The AC-LCM images were imported to Fiji macro to quantify stemness markers' expression (CD24 and CD133). (A) Schematics of altered stemness markers in a normoxic and hypoxic environment. (B) Flow diagram illustrating quantitative measurement of (corrected total cell fluorescence) CTCF for CD24 and CD133 expression by using Fiji macro. (C-D) Representative (AC-LSM) images indicating CD24 or CD133 expression (green), nuclei (blue), F actin (red), cytoplasmic membrane (magenta) of MB231 (C), and TNBC patient-derived cells (D) in a normoxic and hypoxic environment (scale bar, 5 μm). (B) Flow diagram illustrating quantitative measurement of (corrected total cell fluorescence) CTCF for CD24 and CD133 expression by using Fiji macro. (E-F) Quantification of normalized intensity for CD24 and CD133 expression in normoxic and hypoxic conditions on MDA-MB-231 cells (E) and TNBC PDX cells (F).


Fig. 2. Invaded triple-negative breast cancer (TNBC) cells express higher CD24 and CD133 expression in a hypoxic microenvironment. TNBCs (MDA-MB 231 and patient-derived xenograft cells) were cultured for three days in a normoxic and hypoxic environment on 0.4 um polycarbonate membrane inserts. The invaded cells were collected in the bottom chamber, fixed, stained with DAPI, and either CD24 Ab or CD133 Ab and imaged using Airyscan Confocal laser-scanning Microscopy (AC-LSM). AC-LCM images were imported to Fiji macro to quantitate the expression of stemness markers (CD24 and CD133). (A) Schematics of altered stemness marker analysis workflow for invaded tumor cells. (B-C) Quantification of normalized intensity for CD24 (B) and CD133 (C) expression in normoxic and hypoxic conditions on invaded TNBC PDX cells (D). Representative (AC-LSM) images indicating CD24 or CD133 expression (green), nuclei (blue) of TNBC PDX cells in a normoxic and hypoxic environment.


Fig. 3. Dox-induced enhanced stemness expression reduced with synergistic DOX and ATRA combination treatment. TNBC PDx cells were cultured in a normoxic and hypoxic environment for three days. Those cultured cells were treated with either Dox or Dox and ATRA for 72h after that fixed, stained with DAPI, and either CD24 Ab or CD133 Ab, and imaged using Airyscan Confocal laser-scanning Microscopy (AC-LSM). AC-LCM images were imported to Fiji macro to quantitate the expression of stemness markers (CD24 and CD133). (A) Representative (AC-LSM) images indicating CD24 or CD133 expression (green), nuclei (blue), Dox (Red) of TNBC PDX cells in hypoxic environment. The graph represents the quantification of Dox-induced CD24 and CD133 enhanced expression in normoxic and hypoxic conditions on TNBC PDX cells. Dotted red line indicates mean normalized intensity of cells without treatment. (B) The colormap demonstrated Dox and ATRA synergistic concentrations. The graph represents altered CD24 and CD133 expression due to the synergistic effect. Dotted red line indicates mean normalized intensity of cells without treatment.